Protein kinases represent a large family of proteins that play a central role in the regulation of a wide variety of cellular processes. Through regulating an array of signaling pathways, protein kinases control cell metabolism, cell cycle progression, cell proliferation and cell death, differentiation and survival. There are over 500 kinases in the human kinome, and over 150 of these have been shown or are proposed to be involved in the onset and/or progression of various human diseases including inflammatory diseases, cardiovascular diseases, metabolic diseases, neurodegenerative diseases and cancer.
A partial list of such kinases include abl, AATK, ALK, Akt, Axl, bmx, bcr-abl, Blk, Brk, Btk, csk, c-kit, c-Met, c-src, c-fins, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, cRaf1, CSF1R, CSK, DDR1, DDR2, EPHA, EPHB, EGFR, ErbB2, ErbB3, ErbB4, Erk, Fak, fes, FER, FGFR1, FGFR2, FGFR3, FGFR3, FGFR4, FGFR5, Fgr, flt-1, Fps, Frk, Fyn, GSG2, GSK, Hck, LLK, INSRR, IRAK4, ITK, IGF-1R, INS-R, Jak, KSR1, KDR, LMTK2, LMTK3, LTK, Lck, Lyn, MATK, MERTK (Mer), MLTK, MST1R (Ron), MUSK, NPR1, NTRK, MEK, MET, PLK4, PTK, p38, PDGFR, PIK, PKC, PYK2, RET, ROR1, ROR2, RYK, ros, SGK493, SRC, SRMS, STYK1, SYK, TEC, TEK, TEX14, TNK1, TNK2, TNNI3K, TXK, TYK2, Tyro-3, tie, tie2, TRK, Yes, and Zap 70.
Protein tyrosine kinases are a subclass of protein kinase. They also may be classified as growth factor receptor (e.g., Axl, Mer, c-Met (HGFR), Ron, EGFR, PDGFR and FGFR) or non-receptor (e.g., c-src and bcr-abl) kinases. Receptor tyrosine kinases are transmembrane proteins that possess an extracellular binding domain for growth factors, a transmembrane domain, and an intracellular portion that functions as a kinase to phosphorylate a specific tyrosine residue in proteins. Abnormal expression or activity of protein kinases has been directly implicated in the pathogenesis of myriad human cancers.
Axl and Mer are both members of the TAM receptor family, which also includes Tyro3. All three are activated by a common ligand, Growth Arrest-specific protein 6 (Gas6), and they ordinarily play an embryonic developmental role in cell survival, migration and differentiation. The TAM receptors are characterized by a combination of two immunoglobulin-like domains and dual fibronectin type III repeats in the extracellular region and a cytoplasmic kinase domain (Trevor et al., “The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro3/Axl family of receptor tyrosine kinases” Cell, 1995, 80, 661-670; Varnum et al., “Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6” Nature, 1995, 373, 623-626).
Axl signaling is required to maintain EMT-associated features including invasiveness and metastasis (Linger et al., “TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer” Adv. Cancer Res., 2008, 100, 35-83). Axl overexpression and signaling has been implicated in several human malignancies, such as colon, breast, glioma, thyroid, gastric, melanoma, lung cancer, and in renal cell carcinoma (RCC). A more detailed role of Axl biology has been proven in glioma, where loss of Axl signaling diminished glioma tumor growth, and in breast cancer, where Axl drive cell migration, tube formation, neovascularization and tumor growth. Axl has been shown to play multiple roles in tumorigenesis and that therapeutic antibodies against Axl may block Axl functions not only in malignant tumor cells but also in the tumor stroma. The additive effect of Axl inhibition with anti-VEGF suggests that blocking Axl function could be an effective approach for enhancing antiangiogenic therapy (Li et al., “Axl as a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis and angiogenesis” oncogene, 2009, 28, 3442-3455; and Linger et al., “TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer” Adv. Cancer Res., 2008, 100, 35-83).
High levels of Axl expression have been correlated with poor survival in many types of cancer, including breast cancer (Christine et al., “Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival” Proc. Natl. Acad. Sci. USA, 2010, 107(3), 1124-1129), acute myeloid leukemia (Amer. Soc. Hematol. Annual Meeting, San Diego 2011), glioblastoma multiforme (Markus et al., “Axl and growth arrest-specific gene 6 are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme” Clin. Cancer Res., 2008, 14, 130-138) and osteosarcoma (Han et al., “Gas6/Axl mediates tumor cell apoptosis, migration and invasion and predicts the clinical outcome of osteosarcoma patients” Biochem. Biophys. Res. Commun., 2013, 435(3), 493-500). In addition, activation of Axl kinase has been identified as one mechanism by which lung cancers can develop resistance to therapies targeting EGFR, such as Tarceva (erlotinib) (Zhang et al., “Activation of the Axl kinase causes resistance to EGFR-targeted therapy in lung cancer” Nat. Genet., 2012, 44(8), 852-860).
Mer expression correlates with disease progression. It has been found that Mer expression was high in metastatic melanomas (Jennifer et al., “MERTK receptor tyrosine kinase is a therapeutic target in melanoma” J. Clin. Invest., 2013, 123(5), 2257-2267), and activation of Mer promotes invasion and survival in glioblastoma multiforme (Wang et al., “Mer receptor tyrosine kinase promotes invasion and survival in glioblastoma multiforme” Oncogene, 2013, 32, 872-882). Studies also indicated a role for Mer in acute lymphoblastic leukemia (ALL). Mer is ectopically expressed in at least 50% of pediatric T-cell ALL samples as well as in pre-B ALL samples (Graham et al., “Ectopic expression of the proto-oncogene Mer in pediatric T-cell acute lymphoblastic leukemia” Clin. Cancer Res., 2006, 12(9), 2662-2669). Thus, Mer receptor tyrosine kinase is proposed to be a therapeutic target for various solid or hematological malignancies.
Recently a study showed that Mer and Axl were frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. Abnormal expression and activation of Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicated that Mer and Axl play complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Therefore, inhibition of both Axl and Mer is potentially a therapeutic strategy to target cancer cells (Rachel et al., “Mer or Axl Receptor Tyrosine Kinase inhibition promotes apoptosis, blocks growth, and enhances chemosensitivity of human non-small cell lung cancer” Oncogene, 2013, 32(29), 3420-3431).
c-Met, also referred to as hepatocyte growth factor receptor (HGFR), is expressed predominantly in epithelial cells but has also been identified in endothelial cells, myoblasts, hematopoietic cells and motor neurons. The natural ligand for c-Met is hepatocyte growth factor (HGF), also known as scatter factor (SF). In both embryos and adults, activated c-Met promotes a morphogenetic program, known as invasive growth, which induces cell spreading, the disruption of intercellular contacts, and the migration of cells towards their surroundings (Peschard et al., “From Tpr-Met to Met, tumorigenesis and tubes” oncogene, 2007, 26, 1276-1285; and Christine et al., “MET receptor tyrosine kinase as a therapeutic anticancer target” Cancer Letters, 2009, 280(1), 1-14).
A wide variety of human malignancies exhibit sustained c-Met stimulation, overexpression or mutation, including carcinomas of the breast, liver, lung, ovary, kidney, thyroid, colon, glioblastomas and prostate, etc. c-Met is also implicated in atherosclerosis and lung fibrosis. Invasive growth of certain cancer cells is drastically enhanced by tumor-stromal interactions involving the HGF/c-Met pathway. Thus, extensive evidence that c-Met signaling is involved in the progression and spread of several cancers and an enhanced understanding of its role in disease have generated considerable interest in c-Met as major targets in cancer drug development (Cristina et al., “Molecular cancer therapy: Can our expectation be MET” Eur. J. Cancer, 2008, 44(5) 641-651; and Peruzzi et al., “Targeting the c-Met signaling pathway in cancer” Clin. Cancer Res., 2006, 12(12), 3657-3660).
Ron (MST1R, recepteur d'origine nantais), the other member of the MET family, is a receptor tyrosine kinase for the ligand macrophage-stimulating protein (MSP, also known as MST1, and hepatocyte growth factor-like (HGFL) protein), which is associated with in vitro and in vivo cell dissociation, motility and matrix invasion—all of which are surrogate markers of an aggressive cancer phenotype with metastatic potential. Ron mediates oncogenic phenotypes in lung, thyroid, pancreas, prostate, colon and breast cancer cells and predicts a poor prognosis in human breast cancer. Co-expression of Ron with Met and the induction of Ron expression by HGF-Met signaling have both been described in hepatocellular carcinoma. Furthermore, co-expression of Met and Ron portends a worse prognosis in ovary, breast and bladder cancers. Given Ron and Met signaling redundancy, it is possible that resistance to Met inhibition is mediated by Ron signaling (Catenacci et al., “RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma” Cancer Biol. Ther., 2011, 12(1), 9-46).
The roles of MSP-Ron signaling axis in cancer pathogenesis has also been extensively studied in various model systems. Both in vitro and in vivo evidence has revealed that MSP-Ron signaling is important for the invasive growth of different types of cancers. Aberrant Ron activation, which is induced by overexpression of protein and the generation of oncogenic isoforms and is indicated by the persistent activation of multi-intracellular signaling cascades, occurs in various types of cancers. Ron signaling is also necessary for cancer cell growth and survival. These features render Ron as a drug target for cancer therapy (Yao et al., “MSP-RON signalling in cancer: pathogenesis and therapeutic potential” Nat. Rev. Cancer, 2013, 13(7), 466-481).
It is widely known that cancer cells employ multiple mechanisms to evade tightly regulated cellular processes such as proliferation, apoptosis and senescence. Thus, most tumors can escape from the inhibition of any single kinase. System-wide analysis of tumors identified receptor tyrosine kinase (RTK) coactivation as an important mechanism by which cancer cells achieve chemoresistance. One of the strategies to overcome RTK coactivation may involve therapeutically targeting multiple RTKs simultaneously in order to shut down oncogenic RTK signaling and overcome compensatory mechanisms (Alexander et al., “Receptor tyrosine kinase coactivation metworks in cancer” Cancer Res., 2010, 70, 3857-3860). Anti-tumor approaches in targeting Axl, Mer, c-Met and/or Ron signaling may circumvent the ability of tumor cells to overcome Axl, Mer (MERTK), c-Met (HGFR) and/or Ron (MST1R) inhibition alone and thus may represent improved cancer therapeutics.